scholarly journals Evaluation of the SARS-CoV-2 inactivation efficacy associated with buffers from three kits used on high-throughput RNA extraction platforms

2021 ◽  
Author(s):  
Ruth E Thom ◽  
Lin Eastaugh ◽  
Lyn O'Brien ◽  
David Ulaeto ◽  
James S Findlay ◽  
...  
Keyword(s):  
Heat Treatment ◽  
High Throughput ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Sample Type ◽  
Diagnostic Laboratory ◽  
Viral Pathogen ◽  
Viral Transport ◽  
Viral Transport Medium ◽  
Lysis Buffer

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QiaCube HT and the ThermoFisher Kingfisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type; SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer from the MagMax Pathogen RNA/DNA kit (ThermoFisher), used on the Kingfisher Flex, which included guanidinium isothiocycnate (GITC), a detergent, and isopropanol demonstrated a minimum inactivation efficacy of 1 x 105 TCID50/ml.  An alternative lysis buffer from the MagMax Viral/Pathogen Nucleic Acid kit (Thermofisher) also used on the Kingfisher Flex and the lysis buffer from QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QiaCube HT (both of which contained GITC and a detergent) reduced titres by 1 x 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 minutes, 68 °C) did not completely inactivate the virus, demonstrating a reduction of 1 x 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

scholarly journals Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms

2021 ◽  
Vol 11 ◽  
Author(s):  
Ruth E. Thom ◽  
Lin S. Eastaugh ◽  
Lyn M. O’Brien ◽  
David O. Ulaeto ◽  
James S. Findlay ◽  
...  
Keyword(s):  
Heat Treatment ◽  
High Throughput ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Sample Type ◽  
Diagnostic Laboratory ◽  
Viral Pathogen ◽  
Infectious Dose ◽  
Viral Transport Medium ◽  
Lysis Buffer

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

scholarly journals Improved Sensitivity, Safety, and Rapidity of COVID-19 Tests by Replacing Viral Storage Solution with Lysis Buffer

2020 ◽  
Author(s):  
Oran Erster ◽  
Omer Shkedi ◽  
Gil Benedek ◽  
Eyal Zilber ◽  
Itay Varkovitzky ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Detection Threshold ◽  
Test Tube ◽  
Clinical Samples ◽  
Sample Collection ◽  
Standard Guideline ◽  
Viral Transport Medium ◽  
Two Samples ◽  
Lysis Buffer ◽  
And Control

AbstractConducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic.Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling.We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB).We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

scholarly journals Improved sensitivity, safety, and rapidity of COVID-19 tests by replacing viral storage solution with lysis buffer

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0249149
Author(s):  
Oran Erster ◽  
Omer Shkedi ◽  
Gil Benedek ◽  
Eyal Zilber ◽  
Itay Varkovitzky ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Detection Threshold ◽  
Test Tube ◽  
Clinical Samples ◽  
Sample Collection ◽  
Standard Guideline ◽  
Viral Transport Medium ◽  
Two Samples ◽  
Lysis Buffer ◽  
And Control

Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Evaluation of Chemical Protocols for Inactivating SARS-CoV-2 Infectious Samples

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 624 ◽  
Author(s):  
Boris Pastorino ◽  
Franck Touret ◽  
Magali Gilles ◽  
Lea Luciani ◽  
Xavier de Lamballerie ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Rna Extraction ◽  
Cell Culture Supernatant ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
European Standard ◽  
Lysis Buffer ◽  
Level 2 ◽  
Biosafety Level ◽  
Chaotropic Agents

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 min contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.

scholarly journals Rapid isothermal amplification and portable detection system for SARS-CoV-2

2020 ◽  
Vol 117 (37) ◽  
pp. 22727-22735 ◽  
Author(s):  
Anurup Ganguli ◽  
Ariana Mostafa ◽  
Jacob Berger ◽  
Mehmet Y. Aydin ◽  
Fu Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Alternative Pathway ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Rt Pcr ◽  
Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

scholarly journals Studi Awal Analisis Molekuler Human Papillomavirus dari Apusan Glans dan Batang Penis

2021 ◽  
Vol 9 (4) ◽  
pp. 433
Author(s):  
Maya Savira ◽  
Resty Yuwandari ◽  
Yossi Maryanti ◽  
Rahmat Azhari Kemal ◽  
Donel S
Keyword(s):  
Human Papillomavirus ◽  
Rna Extraction ◽  
Viral Rna ◽  
Transport Medium ◽  
Viral Transport ◽  
Viral Transport Medium

Pria juga dapat mengalami keganasan akibat infeksi Human Papillomavirus (HPV) serta bertindak sebagai reservoir virus. Metode skrining HPV pada wanita telah terstandardisasi, namun belum ada standar metode skrining pada pria di Indonesia. Beberapa studi pada populasi pria di luar negeri menunjukkan potensi sampling pada daerah genitalia eksterna untuk skrining HPV. Tujuan: mengoptimasi metode skrining HPV secara molekuler pada pria. Metode: Responden adalah partner seksual wanita pansien kanker serviks di RSUD Arifin Achmad Provinsi Riau. Apusan dari glans dan batang penis diambil menggunakan nylon-flocked swab yang kemudian dimasukkan ke dalam 350µl viral transport medium terpisah. DNA diisolasi dari sampel yang kemudian dianalisis untuk mendeteksi gen human β-globin dan HPV. Hasil: Optimasi awal menunjukkan gen β-globin dapat terdeteksi dari hasil ekstraksi dengan kit Zeesan Viral RNA Extraction. Pita HPV hasil PCR dengan primer MY09 dan MY11 dapat muncul namun masih tipis. Simpulan: Studi awal ini menunjukkan bahwa apusan glans dan batang penis dapat digunakan untuk deteksi HPV secara molekuler pada pria, namun proses pengambilan sampel, ekstraksi DNA, dan PCR masih perlu dioptimasi.Kata kunci: apusan, glans, HPV, penis

scholarly journals Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

2015 ◽  
Vol 53 (10) ◽  
pp. 3148-3154 ◽  
Author(s):  
Sophie J. Smither ◽  
Simon A. Weller ◽  
Amanda Phelps ◽  
Lin Eastaugh ◽  
Sarah Ngugi ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Clinical Sample ◽  
Rna Extraction ◽  
Heat Treatments ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sample Type ◽  
Viral Inactivation ◽  
Blood Samples ◽  
Diagnostic Samples

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10850% tissue culture infective dose per milliliter [TCID50· ml−1]) and murine blood (EBOV concentration of 1 × 107TCID50· ml−1) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.

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